Soukupová Marie, Zucchini Silvia, Trempat Pascal, Ingusci Selene, Perrier-Biollay Coline, Barbieri Mario, Cattaneo Stefano, Bettegazzi Barbara, Falzoni Simonetta, Berthommé Hervé, Simonato Michele
Department of Neuroscience and Rehabilitation, Section of Pharmacology, University of Ferrara, 44121 Ferrara, Italy.
Laboratory of Technologies for Advanced Therapy (LTTA), Technopole of Ferrara, 44121 Ferrara, Italy.
Mol Ther Methods Clin Dev. 2021 Mar 29;21:399-412. doi: 10.1016/j.omtm.2021.03.020. eCollection 2021 Jun 11.
A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.
开发神经系统疾病基因治疗策略的一个关键因素是合适载体的可用性。目前，最先进的是腺相关载体，它们虽然安全并能确保转基因的长期表达，但其承载能力非常有限。相比之下，基于单纯疱疹病毒的扩增子载体可以容纳大量的外源DNA，但人们对其安全性和长期表达转基因的能力存在担忧。我们旨在使用不同的启动子来调节和延长扩增子诱导的转基因表达动力学，并防止转基因沉默。为了实现后者，我们删除了载体构建过程中产生的细菌DNA序列，并使用富含AT的序列和类似绝缘子的序列（SAm技术）来保护转基因盒。我们使用荧光素酶和绿色荧光蛋白作为报告基因。为了确定转基因表达动力学，我们将载体注射到小鼠海马体中，并对其进行长达6个月的纵向生物发光扫描。为了评估安全性，我们分析了多种损伤标志物并进行了膜片钳电生理实验。所有载体都证明是安全 的，并且我们成功地调节了转基因表达的持续时间，使用SAm技术甚至可以实现稳定、持久的表达。因此，这些扩增子载体是一种灵活、高效且安全的脑内基因递送工具。
